Understanding the interplay between trans-acting proteins and chromatin is fundamental to understanding the regulation of DNA function. This proposal makes use of a structurally defined region of chromatin important in the regulation of yeast cell type to investigate the role of specific factors in establishing nucleosome position. Yeast cell type provides a simple model system for cell type determination in other organisms and is directly relevant to normal developmental processes, oncogenesis, and tumor progression. Repression a cell type specific genes in a cells is mediated through a repressor complex, a2.MCM1. Nucleosomes are precisely and stably positioned in promoter regions adjacent to the a2/MCM1 operator in a cells (+a2), but not in a cells (- a2). Both positioning and repression are disrupted by mutations in the histone H4 amino terminus, indicating that positioned nucleosomes contribute to repression. Several global transcription factors, SSN6, TUP1, SIN4, SIN3, RPD1, and TOP3, are also required for repression, but the molecular functions of these proteins are unknown. Proposed experiments will investigate the location and stability of nucleosomes adjacent to the a2 operator in the absence of each factor to determine which are required for nucleosome positioning. Domains within individual factors will then be mutated to determine if region required for the organization of chromatin are also important for repression. Finally, interactions between these factors and histones will be investigated genetically and biochemically. These studies should greatly increases our understanding of how multiple proteins interact to organize regulatory regions of chromatin. Given the involvement of the H4 amino- terminus in the regulation of other less tractable chromatin domains, these studies may also provide information regarding mechanisms of epigenetic inheritance at the yeast silent mating loci and teleomeres, as well as allele-specific imprinting and dosage compensation observed in higher eukaryotes.